Sequential probing of molecular targets based on pseudo-color barcodes with embedded error correction mechanism

Abstract

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs, or organisms. Pre-designed barcodes are associated with specific molecular targets through sequential hybridization experiments. A pseudo-color based barcoding scheme is described that overcomes the limitations in the previous generation of the technology. The current method can be applied to both in vitro and in situ analysis.

We claim: 
     
         1 . A method of barcoding molecular targets, comprising:
 identifying N molecular targets in a biological sample, wherein the N molecular targets are immobilized;   associating a unique barcode to each molecular target via n sequential barcoding rounds (where n≥2), wherein each barcoding round comprises m serial hybridizations of probes collectively bound to the N molecular targets (where m≥2), wherein each serial hybridization comprises:
 contacting one or more groups of probes to a subset of the N molecular targets, the total number of groups of probes corresponding to the number of molecular targets in the subset, wherein probes in each group comprise one or more binding sequences specifically targeting a molecular target in the subset, wherein each probe is capable of generating at least one detectable visual signal representing binding of the probe to a molecular target in the subset, and wherein probes in the one or more groups generate one or more different detectable visual signals corresponding to the number of molecular targets in the subset; 
 detecting the detectable visual signals that reflect the binding between the one or more groups of probes and the subset of the N molecular targets; and 
 removing the visual signals, when applicable, prior to the next serial hybridization; 
   wherein the unique barcode to each molecular target consists of n components, each component is assigned from S unique symbols, where S is an integer that equal to or greater than   
       
         
           
             
               
                 N 
                 n 
               
               ; 
             
           
         
       
       and
 optionally removing probes between two barcoding rounds. 
 
     
     
         2 . The method of  claim 1 , wherein the detecting the detectable visual signals comprises:
 capturing, for each serial hybridization round, an image of the detectable visual signals that reflect the binding between the one or more groups of probes and the subset of the N molecular targets.   
     
     
         3 . The method of  claim 1 , further comprising:
 generating, for each barcoding round, a composite image by superimposing m images corresponding to the m serial hybridizations, wherein the m images are aligned based on one or more alignment references whose positions remain constant relative to the biological sample.   
     
     
         4 . The method of  claim 1 , further comprising:
 applying Gaussian analysis to super-localize the detectable visual signals in an image.   
     
     
         5 . The method of  claim 1 , further comprising:
 decoding the detectable visual signals in each composite image based on the unique barcodes for the N molecular targets and the S unique symbols.   
     
     
         6 . The method of  claim 1 , further comprising:
 detecting reference visual signals associated with the one or more alignment reference.   
     
     
         7 . The method of  claim 3 , wherein the one or more alignment references comprise one or more selected from the group consisting of an oligonucleotide sequence immobilized on the coverslips and detected by a complementary oligo, a common sequence embedded in all probes, a microscopic object, a metal bead, a gold bead, a polystyrene bead, a PCR handle sequence on a primary binding probe, and combinations thereof. 
     
     
         8 . The method of  claim 1 , wherein the n sequential barcoding rounds includes x round for error correction, where x is an integer equal or greater than 1; and wherein assigning unique barcodes for each of N molecular targets requires S unique symbols, where S is an integer equal or greater than 
       
         
           
             
               
                 N 
                 
                   n 
                   - 
                   x 
                 
               
               . 
             
           
         
       
     
     
         9 . The method of  claim 1 , wherein the biological sample comprises a tissue sample, a cell sample, a cell extract sample, a nucleic acid sample, an RNA transcript sample, a protein sample, an mRNA sample, DNA molecules, protein molecules, RNA and DNA isoform molecules, single nucleotide polymorphism molecules, or combinations thereof. 
     
     
         10 . The method of  claim 1 , further comprising:
 determining a secondary molecular target that are associated with the N molecular targets by contacting the biological sample with molecules specifically binding to the secondary molecular target.   
     
     
         11 . The method of  claim 10 , wherein the secondary molecular target comprises one selected from the group consisting of a RNA binding protein molecule, ribosome, a DNA binding protein molecule, a transcription factor, a chromatin binding protein, a protein binding molecule, a scaffold protein, and combinations thereof. 
     
     
         12 . The method of  claim 1 , wherein probes in the one or more groups of probes further comprise:
 one or more binding sequences each specifically targeting one or more sites within a molecular target in the biological sample; and   n unique readout sequences associated with the one or more binding sequences, wherein, in each barcoding round, only one unique readout sequence is associated with a detectable visual signal for a particular molecular target.   
     
     
         13 . The method of  claim 1 , wherein the one or more binding sequences target multiple different sites within the same molecular target. 
     
     
         14 . The method of  claim 1 , wherein the one or more binding sequences target multiple different sites within different molecular targets. 
     
     
         15 . The method of  claim 1 , wherein each probe comprises one or more of the n unique readout sequences. 
     
     
         16 . The method of  claim 15 , wherein at least one of the n unique readout sequences is located in an overhang sequence directly connected to the binding sequence of a probe. 
     
     
         17 . The method of  claim 16 , wherein at least one of the n unique readout sequences is indirectly connected to the binding sequence of a probe via one or more intermediate molecules. 
     
     
         18 . The method of  claim 17 , wherein the one or more intermediate molecules comprise an RNA bridge probe, a DNA bridge probe, a protein bridge probe, a probe for hybridization chain reaction (HCR), a hairpin nucleic acid probe, an HCR initiator, an HCR polymer, or combinations thereof. 
     
     
         19 . The method of  claim 1 , wherein the one or more binding sequences specifically target one or more non-nucleic acid sites in the molecular target, and wherein the n unique readout sequences comprising nucleic acid sequences that are directly or indirectly connected to the binding sequences of the probes. 
     
     
         20 . The method of  claim 1 , wherein the detectable visual signal is connected to the binding sequence of a probe or an intermediate molecule via a cleavable linker. 
     
     
         21 . The method of  claim 1 , wherein the one or more binding sequences comprises a peptide sequence binding to a specific antigen within a particular molecular target, an aptmer, or click chemistry group. 
     
     
         22 . The method of  claim 1 , wherein the S unique symbols comprise colors, numbers, letters, shapes, or combinations thereof. 
     
     
         23 . The method of  claim 1 , wherein, for each serial hybridization, the one or more groups of probes to a non-overlapping subset of the N molecular targets. 
     
     
         24 . A method of hybridization analysis of binding between labeled probes and molecular targets in a biological sample, comprising:
 generating multiple composite images of labeled probes bound to a plurality of molecular targets in the biological sample, wherein each composite image is generated from a plurality of images of labeled probes collectively bound to the plurality of molecular targets, wherein the plurality of molecular targets are immobilized within the biological sample, and wherein each image of the plurality of images reveals:
 labeled probes bound to a subset of molecular targets within the plurality of molecular targets, wherein the labeled probes comprise one or more groups of probes, the total number of groups of probes corresponding to the number of molecular targets in the subset, wherein probes in each group comprise one or more binding sequences specifically targeting a molecular target in the subset, and wherein each labeled probe is capable of generating a visual signal representing binding of the probe to a molecular target; and 
 one or more reference targets whose positions remain constant in the biological sample for aligning the plurality of images. 
   
     
     
         25 . The method of  claim 24 , wherein the biological sample comprises a tissue sample, a cell sample, a cell extract sample, a nucleic acid sample, an RNA transcript sample, a protein sample, an mRNA sample, DNA molecules, protein molecules, RNA and DNA isoform molecules, single nucleotide polymorphism molecules, or combinations thereof. 
     
     
         26 . The method of  claim 24 , wherein, in each image, the labelled probes bind to a non-overlapping subset of molecular targets within the plurality of molecular targets. 
     
     
         27 . The method of  claim 24 , further comprising:
 contacting the one or more groups of probes with the subset of molecular targets of the plurality of molecular targets;   detecting visual signals that reflect the binding between the one or more groups of probes and molecular targets in the subset; and   removing the visual signals, when applicable, prior to a next round of hybridization of labeled probes binding to a new subset of molecular targets within the plurality of molecular targets.   
     
     
         28 . The method of  claim 24 , further comprising:
 detecting reference visual signals associated with the one or more alignment references.   
     
     
         29 . The method of  claim 24 , further comprising:
 aligning the plurality of images based on the positions of the one or more alignment references.   
     
     
         30 . A sequential hybridization method, comprising:
 identifying a plurality of target genes; and   associating, via sequential hybridization of binding probes to the plurality of target genes, a first plurality of unique codes with the plurality of target genes, wherein each target gene in the plurality of target genes is represented by a unique code in the first plurality of unique codes, wherein the sequential hybridization comprises n rounds of hybridization (where n≥2), and wherein each round of hybridization in n rounds of hybridization comprises:
 contacting the plurality of target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
 a binding sequence that specifically binds a target sequence in a gene in the plurality of target genes, wherein target genes from the plurality of target genes are spatially transfixed from each other, and wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to a target sequence; 
 
 detecting visual signals that reflect the binding between the plurality of binding probes and the plurality of target genes; and 
 removing the visual signals, when applicable, prior to the next round of hybridization; 
   wherein probes used in the n rounds of hybridization are capable of emitting at least F types of detectable visual signals (where F≥2 and F n  is greater than the number of target genes in the plurality of target genes),   wherein a unique code in the first plurality of unique codes for a target gene consists of n components,   wherein each component is determined by visual signals that reflect the binding between binding probes and the target gene during one of the n rounds of hybridization,   wherein the n rounds of hybridization include m error correction round (m≥1),   wherein a second plurality of unique codes for the plurality of target genes is generated after the m error correction round is removed from the n rounds of hybridization, and   wherein each unique code in the second plurality of unique codes consists of (n−m) components and uniquely represents a target gene in the plurality of target genes.   
     
     
         31 . A hybridization method, comprising:
 identifying a plurality of target genes;   performing sequential hybridization of binding probes to the plurality of target genes, wherein the sequential hybridization comprises n rounds of hybridization (where n≥2), and wherein each round of hybridization in n rounds of hybridization comprises:
 contacting the plurality of target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
 a binding sequence that specifically binds a target sequence in a gene in the plurality of target genes, wherein target genes from the plurality of target genes are spatially transfixed from each other, and wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to a target sequence; 
 
 detecting visual signals that reflect the binding between the plurality of binding probes and the plurality of target genes, wherein each target gene in the plurality of target genes is represented by visual signals that are unique for the target gene, and wherein probes used in the n rounds of hybridization are capable of emitting at least F types of detectable visual signals (where F≥2, and F n  is greater than the number of target genes in the plurality of target genes); and 
 removing the visual signals, when applicable, prior to the next round of hybridization; and 
   performing serial hybridizations against one or more serial target genes, wherein the expression level of each serial target gene is above a predetermined threshold value, wherein each serial hybridization comprises:
 contacting the one or more serial target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
 a binding sequence that specifically binds a target sequence in a serial target gene in the one or more serial target genes, wherein one or more serial target genes are spatially transfixed from each other, 
 wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to the target sequence, and wherein probes binding to target sequences in the same serial target gene emit the same detectable visual signals; and 
 
 detecting visual signals that reflect the binding between the plurality of binding probes and the one or more serial target gene. 
   
     
     
         32 . The sequential hybridization method of  claim 30 , wherein each component of a n-component unique code in the first plurality of unique codes is assigned a numerical value that corresponds to one of the at least F types of detectable visual signals; and wherein at least one component of the n-component unique code is determined based on the numerical values of all or some of the other n−1 components. 
     
     
         33 . The sequential hybridization method of  claim 30 , wherein the n-component unique code is determined as: 
       
         
           
             
               
                 { 
                 
                   
                     j 
                     1 
                   
                   , 
                   
                     j 
                     2 
                   
                   , 
                   
                     … 
                     ⁢ 
                         
                     
                       ( 
                       
                         
                           
                             a 
                             1 
                           
                           * 
                           
                             j 
                             1 
                           
                         
                         + 
                         
                           
                             a 
                             2 
                           
                           * 
                           
                             j 
                             2 
                           
                           ⁢ 
                               
                           … 
                         
                         + 
                         
                           
                             a 
                             n 
                           
                           * 
                           
                             j 
                             n 
                           
                         
                         + 
                         C 
                       
                       ) 
                     
                     ⁢ 
                     mod 
                     ⁢ 
                        
                     F 
                   
                   , 
                   … 
                       
                   , 
                   
                     j 
                     n 
                   
                 
                 } 
               
               , 
             
           
         
         wherein j 1  is a numerical value that corresponds the detectable visual signals used in the first round of hybridization, j 2  is a numerical value that corresponds the detectable visual signals used in the second round of hybridization, and j 1  is a numerical value that corresponds the detectable visual signals used in the nth round of hybridization; and 
         wherein j 1 , j 2 , . . . j n , a 1 , a 2 , . . . a n  and n are none zero integers and C is an integer.